Results: 5

Continuous ATF3 accumulation after UV irradiation in CS cells. (A–D) Western blots showing ATF3 expression 24 h after irradiation with 0, 2, 4, 6, 8, 10, and 20 J/m2 UV-C in CS1AN+CSBwt (A), CS1AN (B), CS1AN+Q678E (C), and CS1AN+Q942E (D) cells. (E–H) Western blots determining ATF3 accumulation in untreated cells and 2, 4, 8, 12, 16, and 24 h after exposure to 10 J/m2 UV-C in denoted cell lines. (I–L) ChIP determining Pol II, TFIIB, and ATF3 enrichment at the DHFR CRE/ATF site after exposure to 10 J/m2 UV-C in denoted cell lines. (M–P) ChIP at the DHFR core promoter. (Q–T) ChIP determining Pol II, TFIIB, ATF3, and CSB enrichment at the GADD45 promoter after exposure to 10 J/m2 UV-C in denoted cell lines. A schematic diagram of location of primers used in ChIP experiments is presented at the top of each set of ChIP. All the results are presented as percentage to input giving the respective percentage of enrichment in comparison with chromatin input. Each point represents the average of three real-time PCR reactions in three independent experiments.

α-Amanitin treatment mimics UV-induced stress and causes continued ATF3 accumulation. (A) Western blot with antibody against ATF3 and β-actin as loading control in CS1AN+CSBwt cells incubated with 10 μg/mL α-amanitin for 1 h. Cells were collected at indicated time points after treatment. The genes that are listed from the top to the bottom at the right of Fig. 3 A and B are shown from left to right in each histogram. (B) Quantitative RT-PCR analysis of direct ATF3 target genes (shown in Fig. S1H and Table S2) in CS1AN+CSBwt cells: (i) 24 h after UV-C irradiation (10 J/m2) and (ii) 24 h after administration with 10 μg/mL α-amanitin for 1 h. (C and D) ChIP assay showing the enrichment of Pol II, CSB, and ATF3 at the DHFR promoter at 0, 4, 8, and 24 h after UV (C) or α-amanitin (D) treatment. All results are presented as fold recruitment, which represents the ratio of the value obtained at each time point relative to that of the untreated cells at time t = 0. Each point represents the average of three real-time PCR reactions of three independent experiments.

siRNA-mediated ATF3 down-regulation abolishes the repression of ATF3-dependent genes in CS cells. (A) Western blot analysis of ATF3 protein in CS1AN cells transfected with siCtrl or siATF3 constructs and harvested at different time points after UV-C treatment. (B–E) ChIP experiments showing enrichment of ATF3 on the DHFR CRE/ATF site (B), Pol II on the DHFR core promoter (C), H3K4me2 (D), and acetylated histone H4 on the DHFR core promoter (E) after exposure to 10-J/m2 UV-C in CS1AN+CSBwt, CS1AN+siCtrl, and CS1AN+siATF3 cells. (F) ChIP assay on the DHFR core promoter in CS1AN+CSBwt and CS1AN cell lines showing the stable presence of HDAC1 over a time course of 24 h after UV-C (10 J/m2) treatment in CS1AN and CSi1AN+CSBwt cells. (G and H) Luciferase assay in untreated CS1AN, CS1AN+CSBwt, CS1AN+Q678E, and CS1AN+Q942E cells and 4 and 24 h after 10-J/m2 UV-C irradiation. These cells were transfected with luciferase plasmid with (G) or without (H) a CRE/ATF site in front of the SV40 promoter. All results are presented as fold recruitment, which represents the ratio of the value obtained at each time point relative to that of the untreated cells at time t = 0. Each point represents the average of three real-time PCR reactions of three independent experiments.

ATF3 binding to CRE/ATF sites and subsequent transcriptional repression of target genes. (A) Quantitative RT-PCR analysis of the ATF3 target genes (shown in Fig. S1H and Table S2) in CS1AN+CSBwt cells, CS1AN cells, CS1AN cells transfected with either siCtrl or siATF3, and AS548 cells (19) 24 h after 10-J/m2 UV-C treatment. (B) Quantitative RT-PCR analysis of the newly identified ATF3 target genes in wild-type (CRL-2097) and AS548 neuronal cultures 24 h after treatment with 10-J/m2 UV-C. Cells were harvested at the 0- and 24-h time points after UV-C treatment. Gene expression at 24 h was normalized to 0 h. (C–F) ChIP assays showing enrichment of ATF3 binding at CRE/ATF sites of selected gene promoters. CS1AN (C), CS1AN+CSBwt (D), XPA (XP12RO) (E), and XPC (GM14867) (F) cells were treated with 10 J/m2-UV-C and harvested at different time points within 24 h. The genes that are named from the top to the bottom at the right in A and B are shown from left to right in each histogram. All the results are presented as fold recruitment, the ratio of the value obtained at each time point relative to that of the untreated cells at time t = 0. Each point represents the average of three real-time PCR reactions of three independent experiments.

Arrest of RNA synthesis and induction of IEGs upon genotoxic stress in CS cells. (A) Schematic diagram of CSB secondary structure showing helicase motifs I–VI and the acidic and NTB domains. Cell lines used were transfected with pcDNA3.1 plasmid carrying point mutations with the denoted locations. (B) UV survival assay showing cell density 4 d after exposure to 0–10 J/m2 UV-C radiation. (C and D) DHFR mRNA (C) and GADD45 mRNA (D) after 10-J/m2 UV-C irradiation. Graphs show the average of three independent experiments. (E–G) A quantitative RT-PCR analysis of the set of IEG in CS1AN+CSBwt and CS1AN cells treated with 10 J/m2 of UV-C or 10 μg/mL of α-amanitin as indicated and harvested at different time points after UV-C irradiation. Genes listed from the top to the bottom at the right of G are shown from left to right in each histogram. ATP7A, ATPase, Cu++ transporting, alpha polypeptide DLD, Dihydrolipoamide dehydrogenase; NBN, Nibrin; RAB3GAP2, RAB3 GTPase activating protein subunit 2;RAD50, RAD50 homolog (S. cerevisiae). Other gene symbols are defined in the text. All results are presented as the ratio of the value obtained at each time point relative to that of the untreated cells at time t = 0. Each point represents the average of three independent experiments that were performed in triplicate.